Successful fat-only whole breast reconstruction using cultured mature adipocytes and conditioned medium containing MCP-1

A mastectomy is a curative treatment for breast cancer. It causes breast and soft tissue deficits, resulting in a chest with poor vascularity. Autologous tissue breast reconstruction is commonly associated with donor site morbidity. Breast implants are another reconstruction alternative, but they are associated with infection, rupture, and the need for replacement. Autologous aspirated fat grafting has appeared as an ideal breast reconstruction method, but low graft viability and high resorption remain as the main shortcomings. We developed a novel method for fat-only grafts using cultured mature adipocytes (CMAs) mixed with their condition medium. Twenty-five mastectomy patients, aged 32–72 years, received a mixed grafting of CMAs, MCP1-containing condition medium, and fat grafts for total breast reconstruction. In follow-up periods of 24–75 months, MRI analysis showed full thickness fat-engraftment. The cell proliferation marker Ki67 was negative in post-transplant biopsy specimens from all patients. Aesthetic full breast morphology was achieved, patient satisfaction was evaluated 1 year and 3–6 years after surgery. All grafts were confirmed safe, demonstrating high reliability and long-term sustainability.

www.nature.com/scientificreports/most important component of adipose tissue and are deeply involved in the regulation of adipose formation [26][27][28] .It has been reported that maturation is impaired in the fat of SIRT1-deficient mice.This was thought to be due to impaired angiogenesis as well as fewer intra-adipose macrophages.Intravenous injection of a reagent that inhibits macrophages (clodronate liposomes) in normal mice has been shown to reduce macrophages in adipose tissue and inhibit angioplasty 28 .This indicates that macrophages are essential cells for the complete formation of adipose tissue.
In addition, MCP-1 has been reported to be important for increasing macrophage infiltration into adipose tissue [29][30][31] .This has been demonstrated by the fact that MCP-1 (CCL2) produced in adipose tissue attracts macrophages to tissues in mice lacking the CCR2 receptor 31 .Thus, we decided to use CMAs, which secrete high concentrations of MCP-1 and were expected to further enhance angiogenic and adipogenic potential.
Since 2010, 67 partial breast reconstructions and breast augmentations have been performed with a mixture of CMAs, MCP1-containing conditioned medium, and fat grafts (hereafter referred as CMAM-FGs).The fat engraftment rate of CMAM-FGs was over 90%, and no adverse events occurred.Therefore, CMAM-FGs were applied to whole breast reconstruction after mastectomy, where the efficacy and safety of the treatment were evaluated.

Human mature adipocyte culture
The subjects of this study were 25 patients who had previously undergone a mastectomy.Approximately 3 cc of microfat was harvested from the patient's lower abdomen by subcutaneous aspiration (Fig. 1A).Human mature adipocytes (2-3 × 10 6 cells) were isolated from 2.8 ± 0.9 cc and were cultured at a density of approximately 4 to 5 × 10 4 / cm 2 .The total number of transplanted cells in the clinical application was 884.20 × 10 7 , with a mean value of 17.00 × 10 7 ± 3.09 × 10 7 .(The total number of transplanted cells in the first application was 395.02 × 10 7 with a mean of 15.80 × 10 7 ± 2.33 × 10 7 , the total number of transplanted cells in the second application was 446.28 × 10 7 with a mean of 17.85 × 10 7 ± 3.31 × 10 7 , and the third application was only performed in two patients, bringing the total number of transplanted cells to 42.90 × 10 7 with a mean of 21.45 × 10 7 ± 0).
Cultured mature adipocytes retained a large amount of fat granules that appeared light in phase contrast micrographs (Fig. 1B).Adipocytes were specifically stained red with Sudan III, indicating that mature adipocytes incorporated fat granules (Fig. 1C).

Cultured mature adipocyte aging examination in vitro
A population doubling level (PDL) cell aging test was conducted to confirm the safety of the CMAs from six randomly chosen patients.It was confirmed that CMAs were cultured until they stopped growing (PDL: 55.84 ± 17.63) due to cell aging (61-102 days; 75.67 ± 18.00) (Fig. 4).The results showed that CMAs proceeded to terminal differentiation and stopped proliferating, so the possibility of malignant change (tumorigenesis) was not observed.

Immunofluorescence and immunohistochemical analysis of regenerated fat after the CMAM-FGs in-vivo
Six months to 2 years after CMAM-FGs were transplanted into the chest by injection, biopsy specimens were stained using anti-perilipin fluorescent immunostaining, which is expressed on the cell membrane of adipocytes (Fig. 5A).The results showed that adipose tissue was formed throughout the reconstructed area.After transplantation, the reconstructed fat was rich in macrophages, which were positive in anti-CD68 staining (Fig. 5B).Anti-CD31 and CD34 immunostaining showed many blood vessels in the regenerated tissue (Fig. 5C,D).Cell proliferation marker Ki67 (Fig. 5E), an important molecular target in the diagnosis of cancer 33 , was negative in the biopsy specimens of all patients. .MCP-1 (16.15 ± 2.65 ng/ml) was produced in large quantities, followed by GRO (8.97 ± 3.20 ng/ml), IL-8 (7.09 ± 1.17 ng/ml), and VEGF (1.09 ± 0.43 ng/ml).Production of IL6 (1.17 ± 0.48 ng/ml), MCP-3 (0.29 ± 0.11 ng/ml), fractalkine (0.37 ± 0.07 ng/ml), and G-CSF (0.31 ± 0.13 ng/ml) was also observed.However, only small amounts of other proteins were produced.Indicating values of p < 0.01 as compared to the MCP-1.

Clinical outcomes
The patient summary and clinical results are shown in Table 1.The average engraftment rate of the transplanted CMAM-FGs for the 25 patients was 99.16 ± 4.00%, no postoperative complications were observed in any of the 25 patients.Patients were followed for 2-6 years, and no complications, adverse events or breast cancer recurrences were noted.The graft retention was stable for all patients, and no fat absorption was observed in 24 cases.One patient, who did an intensive exercise program three months after the transplantation, developed a hematoma in the reconstructed breast and absorbed 20% fat.Another patient had a small lump palpated in her reconstructed breast, which was removed and a small cyst found with no evidence of malignancy.

Results of MRI image evaluation and patient assessment
The 25 patients treated were examined with MRI imaging annually to evaluate safety.The reconstructed breast after mastectomy showed fat regeneration across the full thickness (

Discussion
Successful breast reconstruction using only fat after mastectomy requires a high survival rate and long-term graft retention.Although various advantages of fat grafting have been reported in breast reconstruction, resorption of the fat graft remains an important clinical challenge that needs to be resolved [10][11][12][13][14][15] .In general, healthy adipose tissue survives because it is supported by a rich vascular network that nourishes it.Naturally, if there are few blood vessels, the adipose tissue will be malnourished and absorbed.Blood vessels in aspirated fat grafts are crushed by liposuction, so there is no blood flow to the site itself.As a result, the grafts are likely to become necrotic and absorbed after transplantation.Therefore, we thought that the addition of a factor (culture medium) that promotes angiogenesis of fat grafts would improve the fat engraftment rate.
Macrophages are also an important component of adipose tissue and play a key role in angiogenesis and adipogenesis [26][27][28] .It has also been demonstrated that MCP-1 produced in adipose tissue attracts macrophages to the tissue, and MCP-1 has been reported to be important for increasing macrophage infiltration into adipose tissue [29][30][31][33][34][35] . MCP-1 CCL2), which is produced in adipose tissue, has been demonstrated to attract macrophages to the tissue in mice lacking the CCR2 receptor and by using drugs that inhibit CCR2 signaling 28 .www.nature.com/scientificreports/ Futhermore, it has been reported that MCP-1 directly binds to human vascular endothelial cells expressing the CCR2 receptor to promote angiogenesis 31 .We found that the condition medium (CM) secreted by CMAs contained a large amount of MCP-1 (16,000 pg/ ml), which attracted and mobilized macrophages in the transplanted fat.This was evidenced by the abundant expression of CD68 in the regenerated fat tissue, demonstrating the presence of macrophages.In addition, since CD31 and CD34 are expressed in the blood vessels in regenerated fat tissue, it can be said that the sequence of fat tissue regeneration, macrophage recruitment, and angiogenesis was induced, which is useful for maintaining the fat formation.
Furthermore, immunostaining of CMAs showed that they contained VEGFR2-positive vascular endothelial progenitor cells and PDGFR-β-positive preadipocytes.PDGFR-β in vascular endothelial cells has been shown to affect angiogenesis 36 .Blocking VEGF signaling with anti-VEGFR2 antibodies prevent adipogenesis, indicating that VEGFR2-positive cells form the final angiogenic adipose 35 and demonstrating that VEGFR-2-positive cells are required for angiogenesis in transplanted adipose tissue.Because CMAM-FGs contain large amounts of MCP-1 and the vascular progenitor cells mentioned above, we concluded that the vascular network is rapidly restored and macrophages are subsequently mobilized, promoting adipogenesis and resulting in long-term adipose tissue survival.
CMAs secrete the most MCP-1, followed by IL8, IL-6 and VEGF, which are known to be involved in angiogenesis [37][38][39][40][41] .The highest secretion of MCP-1 by our CMAs, which is involved in adipogenesis, may be useful for adipogenesis after the transplantation.CMAs also secrete GRO.GRO is present in normal breast adipose tissue 42 , it is reported to be increased in serum level of obese women and it is associated with adiposity 43 .
In addition, CMAs express very little TNF-α, IL-1β, and INF-γ.Therefore, the mature adipocyte-containing composition obtained by the above method, our CMAM-FGs, have a low potential for causing inflammation and is suitable for adipose tissue maintenance after the transplantation.
Next, we compared ASCs cultured from the SVF fraction without adipocytes with our CMAs, noting that Zwierzina et al. classified SVF cells in human white adipose tissue into three types, all of which also express the mesenchymal stem cell marker CD90 at higher levels as a common feature 44 .Our results show that CMAs cells express approximately tenfold higher levels of CD90 than SVF cells.Although it is possible that SVF could not be completely eliminated from CMAs because they were not enzyme-treated, it is likely that the cells were less damaged; the mixture of adipocytes and SVF may have enhanced their expression compared with SVF-only cells.We also believe that this is because SVF-only eliminates the influence of adipocytes.All of the above indicates that CMA is a distinct cell population from SVF.In addition, CMAs were clearly shown by immunostaining to contain a mixture of both adipose progenitor cells and vascular endothelial progenitor cells, and this mixture may have contributed to the high rate of adipose tissue engraftment and maintenance after transplantation.
It should also be noted that an important factor contributing to the high post-transplantation engraftment rate is the high amount of angiogenic factors secreted by the CMAs, a group of factors that promote adipose tissue retention.Among these, MCP-1 (CCL2), which is released in the largest amount, is essential for complete adipose tissue remodeling because it induces not only angiogenesis but also macrophages necessary for adipose tissue.
We believe that our CMAs cells have achieved long-term survival of transplanted adipose tissue, which is different from conventional adipogenesis, due to the above-mentioned factors.
Cellular senescence studies conducted on six of the 25 patients also showed that the CMAs could be cultured until they stopped growing due to cell aging (PDL: 36.8-84.2,55.84 ± 17.63, 61-102 days: 75.67 ± 18.00) and had no capacity for sustained cell proliferation or transformation.In addition, the biopsy specimens of all patients after transplantation were negative in testing for Ki67, a cell proliferation marker, suggesting that CMAs is safe.
In a meta-analysis of 21 studies, the estimated number of transplants required to complete whole breast reconstruction was inconsistent, ranging from 2 to 7 sessions 11,15 .In a recent report of 22 cases of total breast reconstruction with fat grafting, 11 cases (50%) abandoned the use of fat only reconstruction and converted to breast implants due to inadequate engraftment 45 .This indicates that engraftment of fat grafts has conventionally been unreliable in whole breast reconstruction until now.
We achieved 100% fat-only whole breast reconstruction with only two transplants for unilateral cases and only three transplants for bilateral cases.This indicates that our grafting method is more stable compared to other fat grafting methods.Table 1.Total mastectomy patient summary.The VAS for satisfaction is a horizontal line 10 cm long.At the beginning and at the end, there are two descriptors representing extremes of satisfaction (i.e., no satisfaction and extreme satisfaction).The patients rated their satisfaction by making a vertical mark on the 10 cm line.*In N o 5, hematoma was observed due to excessive exercise 3 months after surgery.The hematoma healed following fine needle aspiration, but the volume decreased by 20% thereafter.Most importantly, the mean engraftment rate was 99.16 ± 4.00%, and graft retention was maintained after 2-6 years, demonstrating greater reliability and sustainability compared to previous fat grafting techniques.
The reconstructed breast, which is made of fatty tissue as in a healthy breast, has a soft and natural aesthetic appearance, leading to high general patient satisfaction (patient satisfaction VAS: 9.24 ± 1.04) 1 year after surgery.Three to six years after surgery a new patient survey was conducted evaluating satisfaction in terms of breast shape, and softness, patients expressed that breast reconstruction had had a positive impact on their daily activities, as well as psychological and general physical wellbeing.Patients were satisfied with the surgical results and indicated the surgery had helped them recover from the loss of their natural breasts.All seven categories of patient satisfaction stayed above 80% after 3-6 years (Fig. 8).The follow-up periods, to date, are 2-6 years with no complications or recurrence of breast cancer.It is also important in terms of patient safety that the cell www.nature.com/scientificreports/proliferation marker Ki67, an important molecular target for cancer diagnosis, was negative in all patient biopsy specimens after transplantation.We plan to continue biopsy testing with patient consent.Further studies with annual MRI examinations are needed to evaluate the long-term clinical efficacy and safety of this procedure.

Patients
This is a retrospective cohort study conducted between December 2015 and December 2022, in which 25 patients with breast cancer who had undergone total mastectomy underwent successful fat-only total breast reconstruction using cultured mature adipocytes and conditioned medium containing MCP-1.This study was approved by the Institutional Review Board of Yanaga Clinic on February, 22, 2015.And then, the study was conducted in accordance with all relevant and applicable governmental and institutional guidelines and regulations, as well as cell therapy guidelines set out by Japanese regenerative medicine law since December, 2015.Yanaga Clinic and Tissue Culture Laboratory, is certified as a Class 2 Regenerative Medicine Cell Processing Facility (registration number: FC7140009; 23 March, 2015) and a Class 2 Regenerative Medicine Provider (registration number for autologous cultured adipocytes transplantation: PB7150005, 11 November, 2015).Clinical outcomes, efficacy, and patient safety were reported as required annually to a government-certified committee on Regenerative Medicine (Gamagori City Hospital Deliberation Committee for Specific Regenerative Medicine as an external committee), the Kyushu office of the Ministry of Health regenerative medicine committee, and finally the Japanese Ministry of Health committee on regenerative medicine.Informed consent was obtained from all participants, and the study protocol conformed to the ethical principles in the Declaration of Helsinki and was approved by the respective institutional review boards.

Preparation of human mature adipocyte culture and condition medium
Approximately 3 cc of fat was harvested from the patient's lower abdomen by subcutaneous aspiration, washed, sterilized with antibiotics, digested with collagenase, finely filtered with cell strainer, and centrifuged to collect only the fat component floating in the supernatant.Blood components containing ASCs + SVF precipitated as a pellet in the lower layer were discarded.The microfat particles (Fig. 1A) were cultured in a medium consisting of 10% autologous serum added to α-MEM medium.

Figure 1 .
Figure 1.Characterization of cultured mature adipocytes in vitro.(A) Images clearly show the state of the microfat.(B) Phase-contrast micrographs show the typical elongated adipocytes contained a large amount of fat granules.(C) Adipocytes are stained red with Sudan III, indicating that mature adipocytes incorporate fat granules into the cells.(D) Expression of VEGFR2 and PDGFRβ in CMAs.CMAs that had been passaged twice were seeded in 1 × 10 4 cells/6 wells (9.6 cm 2 /well).A CMAs-only group and a CMAs + aspirated fat graft mixed group (1:4) were prepared and cultured for 5 days.VEGFR2 and PDGFRβ immunohistochemical staining was conducted.Both groups strongly expressed VEGFR2 and PDGFRβ.Control: Goat primary antibody (negative).

Figure 2 .
Figure 2. Flow cytometry analysis of the expression of cell surface markers related to various stem cells on SVFs (left) and CMAs (right).IC: Isotype control antibodies were used for control samples.CMAs showed higher expression levels of CD44 and CD90 than observed in SVFs (the figure representing format of Contour plots with outliers is shown in Fig.S2).

Figure 5 .
Figure 5. Immunofluorescence and immunohistochemical characterization of regenerated fat 6 months to 2 years after CAM-FG in vivo.(A) Anti-perilipin staining shows the adipocyte membrane (green) with DAPIstained blood vessels and cell nuclei (blue).(B) Immunohistochemical detection for macrophage marker CD68 is shown in regenerated adipose tissue.Immunohistochemical detection of endothelial cell markers CD31(C) and CD34 (D) is shown in vascular remodeling of regenerated adipose tissue.(E) Ki67 is not expressed in regenerated adipose tissue.Representative images from n = 6 replicates.

Figure 6 .
Figure 6.Clinical application of CAM-FG and MRI imaging after transplantation in unilateral case.(A) Prereconstruction view (after mastectomy) (B) Appearance of the breast at 4 years after two CAM-FG applications, 2 years after nipple and areola reconstruction.The nipple and areola were reconstructed using the Yanaga method 49,50 .(C) Transverse MRI image 3 years after transplantation.Parenchyma can be seen on the right side, but the left reconstructed side shows only adipose tissue.The adipose tissue shows up as white, and adipose tissue can be confirmed to have formed in all layers.In addition, blood vessels can be seen in the tissue.No fat necrosis, cysts, or calcifications were observed.Stable adipose tissue formation was observed in this or any of the other cases.

Figure 7 .
Figure 7. Clinical application of CAM-FG and MRI imaging after transplantation in bilateral mastectomy.(A) Pre-reconstruction view (after mastectomy).(B) Appearance of the breast at 3 years after 2 applications of CAM-FG.The nipple and areola were reconstructed using the Yanaga method 47,48 .(C) Transverse MRI image 2 years after transplantation.No fat necrosis, cysts, or calcifications were observed on either reconstructed side.Stable adipose tissue formation was observed.